*3.6. Exoproteome Components Identified in the L. infantum Experimental Proteome*

*Leishmania*-secreted molecules and exosomes are particularly relevant for infection establishment because parasites and exosomes are co-egested during the insect blood meal [60]. A detailed analysis of the *L. infantum* secreted proteins (exoproteome) was carried out by Santarem et al. [35]. These authors found that the proteome profiles were distinct depending on the metabolic stage of the parasites (logarithmic or stationary phase promastigotes). The number of distinct proteins identified in that study was 297, and around 90% of them were also identified in the proteome reported here. In another outstanding study, Atayde et al. [61] analyzed the proteomic composition of *L. infantum* exosomes and extracellular vesicles that were directly isolated from the sand fly midgut. Table 5 lists proteins commonly present in exosome preparations; all of them were identified in the *L. infantum* experimental proteome reported here.


**Table 5.** Common components of *Leishmania* exosomes identified in this *L. infantum* proteome study.

#### *3.7. Other Relevant Proteins Identified in the L. infantum Promastigote Proteome*

Proteins with a high molecular weight (HMW) represent a challenge for mass spectrometry-based assays, as they are usually underrepresented in protein extracts used for proteomic analysis. To overcome this limitation, Brotherton et al. [63] optimized extraction protocols to enrich HMW proteins and membrane proteins in *L. infantum* promastigotes and amastigotes. In our study, we confirmed the presence of tryptic and/or chymotryptic peptides from 35 HMW proteins with a molecular weight (MW) higher than 200 kDa (Supplementary File, Table S9). Among them, the identification of a calpain-like cysteine peptidase was remarkable, as it had an estimated MW of around 700 kDa (LINF\_270010200) and was identified by 124 unique peptides, thus covering 24% of the amino acid sequence.

The flagellum is a characteristic organelle of *Leishmania* that confers motility to the parasite in the promastigote stage, during which this structure is particularly prominent. In a recent publication, an exhaustive structural and functional characterization of the *L. mexicana* promastigote flagellum was reported [64]. In that study, flagella preparations were analyzed by proteomics, and this allowed for the identification of 701 unique proteins for this organelle. Orthologues to around 400 flagellum-specific proteins were identified in the *L. infantum* proteome described here. More importantly, most of the proteins relevant for flagellum assembly and motility in *L. mexicana* promastigotes [64] were identified in the experimental proteome of *L. infantum* promastigotes (Supplementary File, Table S10).
